| Background and objectiveLeft ventricular noncompaction(LVNC)is a congenital cardiomyopathy characterized by an excessive trabecular meshwork of deep intertrabecular recesses within the ventricular myocardium,in which heart failure is one of the most common clinical consequences.However,the mechanism underlying LVNC associated heart failure remains poorly understood.Sorbin and SH3 domain containing protein 2(SORBS2)at the highest levels in the heart was reported to function in cytoskeletal organization,cell adhesion,and signaling pathways.In our study,elevated myocardial SORBS2 was observed in LVNC patients.In view of the abundant content and vital function of SORBS2 in the heart,we hypothesized that elevated SORBS2 might be involved in the development and progression of heart failure in LVNC.MethodsUsing protein mass spectrometry analysis of left ventricular noncompaction(LVNC),arrhythmogenic right ventricular cardiomyopathy(ARVC)and hypertrophic cardiomyopathy(HCM)and normal myocardial tissue(NC).Through analysis and comparison,45 proteins specifically up-regulated only in LVNC were screened out,Sorbin And SH3 Domain Containing 2(SORBS2)specifically up-regulated in LVNC hearts without changes in ARVC,HCM and NC.First,we performed experiments in human myocardial tissue:SORBS2,which was only elevated in LVNC,was verified by western blotting and immunohistochemistry.In addition,in LVNC and normal myocardium,immunofluorescence staining was used to detect the location and expression of ?-tubulin and Junctophilin 2(JP 2),as well as western blotting was used to detect the expression of ?-tubulin and JP 2.Immunofluorescence staining and immunoprecipitation were used to study the effect that the co-localization and interaction between ?-tubulin and SORBS2.Then we conducted in vivo experiments:we used the human embryonic stem cells(hESC)derived cardiomyocytes(hESC-CMs),we analyzed the function and structure of SORBS2 overexpressing human embryonic stem cell derived cardiomyocytes via immunoblotting,immunohistochemistry,immunofluorescence,and confocal Ca2+imaging.We observed the expression of ?-tubulin and whether ?-tubulin had interaction with SORBS2 after successfully transfected lentivirus of SORBS2 in hESC-CMs.Functional experiment,using CardioExcyte 96 and calcium imaging monitoring the changeable of calcium fluorescence intensity in SORBS2 overexpression in hESC-CMs compared to the control group.Finally,we performed in vivo experiment on 8-week wild-type C57 mice:we conducted in vivo experiments wherein the heart tissues were injected with an AAV 9 vector to overexpress SORBS2.Through western blotting,immunohistochemistry and immunofluorescence experiments,we verified whether the SORBS2 overexpression mice were successfully constructed.The transfection efficiency of adeno-associated AAV 9 vector was calculated by fluorescent staining flag label.Calcium spark experiment was conducted on single cell of isolated myocardium to observe the changes of calcium ion fluorescence intensity.The effects of overexpression of SORBS2 on heart function and morphology of mice were observed by echocardiography.And we tested followed by analysis using echocardiography,functional and morphological changes were observed by echocardiography and masson trichrome,T-tubule analysis and Ca2+ imaging,staining in mice whose myocardium overexpressing SORBS2.ResultsThe results showed that the expression of SORBS2 and ?-tubulin in LVNC myocardial tissue was significantly increased,and there was a co-localization and interaction between SORBS2 and ?-tubulin.We also observed that in the myocardial tissue of LVNC patients,the structural integrity of JP 2 was impaired with no changeable expression level of JP 2.In vitro experiments,in human embryonic stem cells-induced cardiomyocytes(hESC-CMs),SORBS2 colocated with ?-tubulin structurally.Overexpression of SORBS2 led to increased expression of ?-tubulin(microtubule densification).Under overexpression of SORBS2,JP 2 was uncoupling with Type two ryanodine receptor(RyR2)in hESC-CMs.The functional results with CardioExcyte 96 and Ca2+indicated that the myocardial cell impedance decreased and the fluorescence intensity of Ca2+decreased.In vivo experiments,adeno-associated virus 9(AAV 9)mediated overexpression of SORBS2 in the ventricle cardiac tissues of mice led to consistent findings with our heart-transplant tissue samples and our hESC-CMs experiments.Specifically,we used AAV 9 to overexpress SORBS2 in the cardiac left ventricle tissues of wild-type mice and assessed various cardiac phenotypes.Overexpression of SORBS2 and normal control group by adeno-associated virus was injected for three weeks through the jugular vein of mice.Confirming the expression and location of SORBS2,the expression of SORBS2 in the mice injected with the AAV 9-cTNT-SORBS2-3 flag virus was increased compared to empty-virus control mice.Immunocytochemistry and immunofluorescence both showed that SORBS2 was localized in the Z-lines of mouse cardiac tissues.The transfection efficiency of the adeno-associated virus was 53.45±3.76%.We also observed the interaction between SORBS2 and ?-tubulin in normal mice heart tissues.Furthermore,masson trichrome staining of the cardiac left ventricle tissues of the SORBS2 overexpression mice revealed obvious fibrosis.Immunofluorescence analysis that we observed microtubule densification and the JP 2 distribution is irregular and disordered in the SORBS2 overexpression mice hearts.We conducted echocardiographic measurements the SORBS2 overexpression mice displayed reduced ejection fraction(EF)and fractional shortening(FS),which were significantly decreased compared to the control group.The calcium spark results showed that the calcium ion strength of the single isolated mouse cardiomyocytes overexpressing SORBS2 decreased.ConclusionsWe identified a novel mechanism through which SORBS2 interacts with ?-tubulin and promotes microtubule densification,eventually effecting JP 2 distribution and T-tubule,potentially contributing to heart failure in LVNC disease. |
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